The Prorocentrales are a unique group of dinophytes based on several apomorphic traits, but species delimitation is challenging within the group. Prorocentrum triestinum was described by Josef Schiller in 1918 as an important bloomforming species from Trieste (Mediterranean, Adriatic Sea) with a conspicuous asymmetric outline and a small, asymmetrically located subapical spine. All subsequent records under this name fail to conform to Schiller’s original description. These inconsistencies have their origin in John Dodge’s 1975 revision of Prorocentrum, which placed Prorocentrum redfieldii, a more symmetrical, slender species with a long apical spine, into synonymy under P. triestinum. To clarify this confusion, we collected samples at the type locality of P. triestinum in Trieste and established a strain that is morphologically consistent with the protologue and suitable for use in epitypification. Morphology and rRNA sequence data of this strain were compared with four new strains identified as P. redfieldii from the Mediterranean Sea and the North Atlantic Ocean. Cells of P. triestinum had an asymmetric outline in lateral view and a small, dorso-subapical spine. These features, which are readily resolved by light microscopy, were distinct from those of the nearly symmetrical and slender cells of P. redfieldii, which had a long, apically located spine. The species are nevertheless closely related and share an identical architecture of the periflagellar area with a distinctive, largely reduced accessory pore together with a very small platelet 7. This apomorphy clearly differentiates both species from other species of Prorocentrum. Both species differ in their primary rRNA sequences, and ITS and LSU sequence differences will enable them to be distinguished in future meta-barcoding studies. The present study demonstrates that P. triestinum and P. redfieldii are distinct species and thus contributes to a reliable biodiversity assessment of Prorocentrum.